DEVELOPMENTAL BIOLOGY 204, 488–507 (1998) ARTICLE NO. DB989003 Follistatin and Noggin Are Excluded from the Zebrafish Organizer Hermann Bauer,* Andrea Meier,* Marc Hild,* Scott Stachel,†,1 Aris Economides,‡ Dennis Hazelett,† Richard M. Harland,† and Matthias Hammerschmidt*,2 *Max-Planck Institut fu¨r Immunbiologie, Stu¨beweg 51, 79108 Freiburg, Germany; †Department of Molecular and Cell Biology, University of California, 401 Barker Hall 3204, Berkeley, California 94720-3204; and ‡Regeneron Pharmaceuticals, Inc., 777 Old Saw Mill River Road, Tarrytown, New York 10591-6707 The patterning activity of the Spemann organizer in early amphibian embryos has been characterized by a number of organizer-specific secreted proteins including Chordin, Noggin, and Follistatin, which all share the same inductive properties. They can neuralize ectoderm and dorsalize ventral mesoderm by blocking the ventralizing signals Bmp2 and Bmp4. In the zebrafish, null mutations in the chordin gene, named chordino, lead to a severe reduction of organizer activity, indicating that Chordino is an essential, but not the only, inductive signal generated by the zebrafish organizer. A second gene required for zebrafish organizer function is mercedes, but the molecular nature of its product is not known as yet. To investigate whether and how Follistatin and Noggin are involved in dorsoventral (D-V) patterning of the zebrafish embryo, we have now isolated and characterized their zebrafish homologues. Overexpression studies demonstrate that both proteins have the same dorsalizing properties as their Xenopus homologues. However, unlike the Xenopus genes, zebrafish follistatin and noggin are not expressed in the organizer region, nor are they linked to the mercedes mutation. Expression of both genes starts at midgastrula stages. While no patterned noggin expression was detectable by in situ hybridization during gastrulation stages, later expression is confined to presumptive cartilage cells in the branchial arches and the neurocranium and to proximal regions of the pectoral fin buds. follistatin transcripts in gastrulating embryos are confined to anterior paraxial regions, which give rise to head mesoderm and the first five somites. The dorsolateral extent of this expression domain is regulated by Bmp2b, Chordino, and Follistatin itself. In addition, transient expression was observed in a subset of cells in the posterior notochord anlage. Later, follistatin is expressed in brain, eyes, and somites. Comparison of the spatiotemporal expression pattern of follistatin and noggin with those of bmp2b and bmp4 and overexpression studies suggest that Noggin and Follistatin may function as Bmp antagonists in later processes of zebrafish development, including late phases of D-V patterning, to refine the early pattern set up by the interaction of Chordino and Bmp2/4. It thus appears that many, but not all, aspects of early dorsoventral patterning are shared among different vertebrate species. © 1998 Academic Press INTRODUCTION step process involving inductive processes driven by differ- ent maternally and zygotically supplied signals (for review, Studies in Amphibia have revealed that axis formation see Harland and Gerhart, 1997). Maternal signals induce and early dorsoventral (D-V) patterning represent a multi- dorsal and ventral mesoderm in the equatorial zone of the early blastula embryo. This coarse early pattern is then Sequence data for this article have been deposited with the refined by zygotic signals. Proteins secreted by the dorsal GenBank Data Library under Accession Nos. AF 084948 (Follista- mesoderm, also called the Spemann organizer (Spemann tin) and AF 084949 (Noggin). and Mangold, 1924), induce neural specification in dorsal 1 Present address: Center for the Application of Molecular Biol- animal regions which would otherwise give rise to epider- ogy to International Agriculture, GPO Box 3200, Canberra ACT 2601, Australia. mal derivatives. In addition, the same signals lead to a 2 To whom correspondence should be addressed. Telephone: 149- dorsalization of initially ventrally specified mesodermal 761-5108 495. Fax: 149-761-5108 358. E-mail: hammerschmid@ cells in lateral regions (see for review, Lemaire and Kodjaba- immunbio.mpg.de. chian, 1996; Harland and Gerhart, 1997). Five proteins 0012-1606/98 $25.00 Copyright © 1998 by Academic Press 488 All rights of reproduction in any form reserved. Follistatin and Noggin in Zebrafish 489 which are expressed in the organizer region of Xenopus described Bmp2/4 antagonists, Follistatin and Noggin? In embryos and which display the inductive properties as- contrast to chordin, no Drosophila follistatin and noggin signed to the Spemann organizer have been identified: homologues have been reported, although Xenopus noggin Chordin (Sasai et al., 1994), Noggin (Smith and Harland, expression was shown to affect D-V patterning by antago- 1992), Follistatin (Hemmati-Brivanlou et al., 1994), Xnr3 nizing Dpp when expressed in Drosophila embryos ( Holley (Smith et al., 1995), and Cerberus (Bouwmeester et al., et al., 1996). In Xenopus, zygotic noggin expression starts at 1996). Among these, Follistatin was originally described as late blastula stages in the dorsal marginal zone and persists an inhibitor of Activin (Nakamura et al., 1989; Hemmati- throughout gastrulation in the prechordal plate and the Brivanlou et al., 1994). In misexpression experiments in presumptive notochord, both derivatives of the Spemann Xenopus embryos, all five proteins can dorsalize ventral organizer. At later stages, noggin expression is initiated at mesoderm (Smith and Harland, 1992; Smith et al., 1993, several new sites, including the roof plate of the neural tube 1995; Sasai et al., 1994; Fainsod et al., 1997; Hsu et al., and skeletogenic cells in the branchial arches (Smith and 1998) and induce neural specification in the absence of Harland, 1992). In the mouse, noggin is expressed in similar mesoderm (Lamb et al., 1993; Hemmati-Brivanlou et al., tissues, namely the node and its axial mesoderm deriva- 1994; Sasai et al., 1995; Hansen et al., 1997). Biochemical tives, as well as the roof plate and condensing cartilage studies indicate that they fulfill their dorsalizing and (McMahon et al., 1998). Mutation of noggin in the mouse neural-inducing activity indirectly via the inhibition of the appears to have no effect on node function, though subse- ventralizing Bone Morphogenetic Proteins (Bmps), mem- quent patterning of both the neural tube and the somites is bers of the TGFb growth factor family which appear to aberrant. function as instructive D-V patterning molecules, deter- Xenopus follistatin expression starts slightly later than mining in a dose-dependent fashion positional identities that of noggin. At the onset of gastrulation, follistatin RNA along the D-V axis of the early Xenopus embryo (Dosch et is detected in a few cells of the Spemann organizer. During al., 1997); Chordin, Noggin, Cerberus, and possibly Follista- gastrulation, follistatin expression continues in the pre- tin bind Bmp2 and Bmp4 on the dorsal side of the embryo, chordal plate and the anterior portion of the notochord thereby preventing binding of the Bmp proteins to their anlage. Beginning at early neurula stages, follistatin expres- receptor (Piccolo et al., 1996; Zimmerman and Harland, sion is initiated at new sites in the head mesoderm; the 1996; Fainsod et al., 1997; Hsu et al., 1998). hypochord; the pronephros; the eyes; the fore-, mid-, and Evidence revealing the requirement of Bmp2b and one of hindbrain; and the midbrain–hindbrain junction (Hemmati- its antagonists, Chordin, during early D-V patterning has Brivanlou et al., 1994). In the mouse, follistatin expression recently come from mutant studies in the zebrafish. Here, pattern is different; embryonic expression of follistatin first large-scale mutant screens (Driever et al., 1996; Haffter et occurs in the primitive streak, followed by expression in al., 1996) have led to the identification of six complemen- head mesoderm, somites, and specific rhombomeres of the tation groups defining six genes required for ventral devel- hindbrain, and later in midbrain and diencephalon (Albano opment (Mullins et al., 1996) and two complementation et al., 1994; Feijen et al., 1994). No expression was reported groups defining two genes required for organizer-dependent in the node, the mouse equivalent of the Spemann organizer dorsal development (Hammerschmidt et al., 1996a). Mo- (Beddington, 1994), or the notochord (Albano et al., 1994; lecular analyses showed that the phenotype of the strongest Feijen et al., 1994). Consistent with these results, follistatin of the dorsalized mutants, swirl, is caused by null muta- knockout mice display later defects, e.g., in muscle and tions in the zebrafish bmp2b gene (Kishimoto et al., 1997; skeleton, and no neural phenotype or defects in early D-V Nikaido et al., 1997; Martı´nez-Barbera´ et al., 1997), while patterning are detected (Matzuk et al., 1995), calling into the ventralization observed in dino mutants is caused by a question whether follistatin is involved in the regulation of null mutation in the zebrafish chordin gene (Schulte- early D-V pattern formation of the mouse embryo. Merker et al., 1997; Fisher et al., 1997). In light of this For a comparative analysis of the roles of Follistatin and relationship, the dino mutant and the zebrafish chordin Noggin in early vertebrate development, we have cloned gene were renamed chordino. Injection studies and double- their zebrafish homologues; examined their spatiotemporal